Critical Evaluation of Embedding Media for Histological Studies of Early Stages of Chick Embryo Development.

A histological examination is an important tool in embryology, developmental biology, and correlated areas. Despite the amount of information available about tissue embedding and different media, there is a lack of information regarding best practices for embryonic tissues. Embryonic tissues are considered fragile structures, usually small in size, and frequently challenging to position correctly in media for the subsequent histological steps. Here, we discuss the embedding media and procedures that provided us with appropriate preservation of tissue and easier orientation of embryos at early development. Fertilized Gallus gallus eggs were incubated for 72 h, collected, fixed, processed, and embedded with paraplast, polyethylene glycol (PEG), or historesin. These resins were compared by the precision of tissue orientation, the preview of the embryos in the blocks, microtomy, contrast in staining, preservation, average time, and cost. Paraplast and PEG did not allow correct embryo orientation, even with agar-gelatin pre-embedded samples. Additionally, structural maintenance was hindered and did not allow detailed morphological assessment, presenting tissue shrinkage and disruption. Historesin provided precise tissue orientation and excellent preservation of structures. Assessing the performance of the embedding media contributes significantly to future developmental research, optimizing the processing of embryo specimens and improving results.

Lead exposure affects cephalic morphogenesis and neural crest cells in Gallus gallus embryo.

The morphogenesis of the head of vertebrates is a process that involves rapid growth and dynamic movements of various cell populations, including the neural crest cells (NCC). These pluripotent cells generated during neurulation have high proliferative and migratory capacity but xenobiotic agents can affect these migratory periods and cause congenital malformations. Lead (Pb) is the most common toxic metal in the environment and a potent teratogen that can affect growth and induce malformations. Despite the known toxic effects of Pb, there is a gap in knowledge about the impact of realistic concentrations of Pb at critical periods of early development. Here, we evaluated mortality, embryonic morphology, NCC migration, and the amount of Pb deposition in chicken embryos after 3 to 4 days of exposure. One of the most interesting observations in this study is that only about 34% of the injected Pb was present in the embryos after 4 days. We observed that exposure to Pb, even under low concentrations, increased mortality and the occurrence of malformations during embryonic development, especially in the cephalic region (CR). Although Pb was found widely distributed in the CR, no relation between its presence and the migration routes of cephalic NCC was observed. But the number of NCC and their migratory distance were reduced. These changes are consistent and explain the morphological anomalies described in this study, which also correlates with the morphofunctional abnormalities reported in the literature. Therefore, this study highlights the concern of exposure to low concentrations of this metal.

Micropollutants impair the survival of Oreochromis niloticus and threat local species from Iguaçu River, Southern of Brazil.

The wastewater contamination of urban rivers is a concern for biodiversity and a consequence from poor urban conservation policies. In the current study, the impact of urban and industrial activities was investigated in Iguaçu river (Southern Brazil) using juvenile Oreochromis niloticus, after trophic and chronic exposure (25, 50 and 100 %), over 81 days. After exposure liver, gills, gonads, brain, muscle, and blood were sampled for chemical, biochemical, histopathological, genotoxic and molecular analyses. Water levels of persistent organic pollutants such as polychlorinated biphenyls, organochlorine pesticides, polycyclic aromatics hydrocarbon (PAHs) and metals were investigated. The redox unbalance, histopathological and increase in vitellogenin expression in fish revealed both the bioavailability of micropollutants and their harmful effects. According to the results, the level of Iguaçu river pollution negatively impacts the health of O. niloticus revealing and highlighting the risk of this pollution exposure to biota and human populations.

Morphological evidence of neurotoxic effects in chicken embryos after exposure to perfluorooctanoic acid (PFOA) and inorganic cadmium.

Perfluorooctanoic acid (PFOA) and cadmium (Cd) are persistent environmental pollutants, bioaccumulative, and capable of crossing the placental barrier. There is evidence of developmental toxicity induced by PFOA and Cd, but their effects on early development are still poorly understood. In the present study, chicken embryos were exposed to PFOA, Cd, and a mixture of PFOA + Cd from blastula stage up to 72 h of incubation. Embryo mortality and morphology were assessed. PFOA and Cd exposure increased mortality and induced malformations, mainly in the cephalic region, the neural tube, and the ventral body wall. After mixed exposure the effect was observed mostly in the neural tube and the cephalic region. However, the mechanism remains unknown. Because of the resemblance between human and chick development, it was possible to correlate the observed alterations in chickens with known human congenital malformations. Therefore, PFOA and Cd are neurotoxic to chicken embryos inducing morphological abnormalities and are possibly neurotoxic to human embryos.

Diffuse sources of contamination in freshwater fish: Detecting effects through active biomonitoring and multi-biomarker approaches.

Aquatic organisms are usually exposed to a mixture of xenobiotics that may exert a large effect even in low concentrations, and when information is obtained exclusively from chemical analyses the prediction of the deleterious effects is potentially hindered. Therefore, the application of complementary monitoring methods is a priority. Here, in addition to chemical analyses, an active biomonitoring study using multiple biomarker responses in Nile tilapia Oreochromis niloticus was conducted to assess the effects of a contamination gradient along four reservoirs in Iguaçu River. Chemical analysis in the muscle showed high levels of metals in fish from the reservoir closest to an industrialized and environmentally degraded area, however fish exposed to all studied reservoirs showed hepatic alterations (necrosis and inflammatory processes). Also, significant variations of biochemical biomarkers were observed with no clear indication of contamination gradient, since an indicative of higher impact was found in an intermediary reservoir, including high concentrations of biliary polycyclic aromatic hydrocarbons (PAHs). However, nuclear morphological alterations (NMA) were less frequent at the same reservoir. Thus, the multi-biomarker approach allied to active biomonitoring is a practical and important tool to assess deleterious effects of contamination in freshwater, providing data for monitoring and conservation protocols.

Environmental risk assessment in five rivers of Parana River basin, Southern Brazil, through biomarkers in Astyanax spp.

In the current study, water quality of five river sites in Parana River basin (Brazil), utilized for public water supply, was assessed through a set of biomarkers in fish Astyanax spp. Population growth and inadequate use of land are challenges to the preservation of biodiversity and resources such as water. Some physicochemical parameters as well as somatic indexes, gills and liver histopathology, genotoxicity, and biochemical biomarkers were evaluated. The highest gonadosomatic index (GSI) and antioxidant parameters (catalase and glutathione S-transferase activities, non-protein thiols), as well as the lowest damage to biomolecules (lipid peroxidation, protein carbonylation, DNA damage) were observed in site 0 (Piava River), which is located at an environmental protected area. Site 1, located in the same river, but downstream site 0 and outside the protection area, presents some level of impact. Fish from site 2 (Antas River), which lack of riparian forest and suffer from silting, presented the highest micronucleus incidence and no melanomacrophages. Differently, individuals from site 3 (Xambrê River) and site 4 (Pinhalzinho River) which receive surface runoff from Umuarama city, urban and industrial sewage, have the highest incidences of liver and gill histopathological alterations, including neoplasia, which indicated the worst health conditions of all sites. In particular, site 4 had high levels of total nitrogen and ammonia, high turbidity, and very low oxygen levels, which indicate important chemical impact. Comparison of the biomarkers in fish allowed classification of the five sites in terms of environmental impact and revealed that sites 3 and 4 had particular poor water quality.

Water quality assessment of the Tubarão River through chemical analysis and biomarkers in the Neotropical fish Geophagus brasiliensis.

The Tubarão River rises in Santa Catarina, Brazil, and has been historically affected by coal mining activities around its springhead. To evaluate its water conditions, an investigation regarding a possible decontamination gradient associated with the increased river flow toward the estuary, as well as the influence of seasonality over this gradient was performed through a series of biomarkers (vitellogenin, comet assay, lipid peroxidation, protein carbonylation, gluthatione, gluthatione S-transferase, acetylcholinesterase, light microscopy in liver, and scanning electron microscopy in gills) and chemical analysis (polycyclic aromatic hydrocarbons (PAHs) in bile and metal analysis in sediment) in the cichlid Geophagus brasiliensis. Two collections (summer and winter) were made in four distinct sites along the river, while sediments were sampled between those seasons. As expected, the contamination linked exclusively to mining activities was not observed, possibly due to punctual inputs of contaminants. The decontamination gradient was not observed, although seasonality seemed to have a critical role in the responses of biomarkers and availability of contaminants. In the summer, the fish presented higher histopathological damages and lower concentrations of PAHs, while in the winter they showed both higher genetic damage and accumulation of PAHs. The Tubarão suffers impacts from diverse activities, representing health risks for wild and human populations.

The distribution of ephrin-B1 and PNA-positive glycoconjugates is correlated with atypical melanoblast migration in Japanese Silky fowl embryos.

Melanoblasts are positively stimulated to migrate in the dorsolateral pathway of the avian embryo by ephrins, but are inhibited by PNA-binding glycoconjugates. We analyzed the potential role of these molecules in the Japanese Silky fowl, which displays intense internal pigmentation. The distribution of ephrin ligands was analyzed using Eph receptor-human Fc fusion proteins. Glycoconjugates were labeled using PNA-FITC. In Japanese Silky embryos, ventral areas, including the anterior- and posterior-half somites, expressed ephrin-B1 in a pattern that correlates with the atypical migratory pathways taken by Japanese Silky melanoblasts. White Leghorn embryos displayed little to no ephrin-Bs in the ventral paths. Conversely, PNA-binding barrier tissues, proposed to prevent melanoblasts from migrating ventrally in White Leghorn, are missing or have significant gaps in Japanese Silky embryos. Thus, studies of a naturally occurring pigmentation mutant confirm that a combination of cues regulates melanoblast migration in the chick embryo.

Curcumin/xanthan-galactomannan hydrogels: rheological analysis and biocompatibility.

Curcumin, a lipophilic compound found in the plant Curcuma longa L., exhibits a wide range of pharmacological activity; however, its therapeutic use has been limited because of its low bioavailability following oral administration. The aim of this study was to evaluate the viscoelastic characteristics and biocompatibility of a curcumin/xanthan:galactomannan hydrogel (X:G) system after topical application on chick embryo chorioallantoic membrane (CAM), a system established with a view toward curcumin nasal or topical pharmaceutical applications or possible administration in cosmetics or foods. A rheological analysis indicated that incorporation of curcumin did not alter the viscoelastic characteristics of the X:G hydrogel, suggesting that there was no change in the structure of the gel network. X:G hydrogels did not induce CAM tissue injury and the curcumin/X:G hydrogel system was also highly biocompatible. We conclude that the X:G hydrogel represents a potential matrix for curcumin formulations.